An ISFET-Based Anion Sensor for the Potentiometric Detection of Organic Acids in Liquid Chromatography

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Sensors and Actuators B 67Ž. 2000 294±299 CORE Metadata, citationwww.elsevier.nl and similarrlocate papersrsensorb at core.ac.uk Provided by Universiteit Twente Repository

An ISFET-based anion sensor for the potentiometric detection of organic acids in liquid chromatography

I. Poels a, R.B.M. Schasfoort b, S. Picioreanu c,1, J. Frank c, G.W.K. van Dedem c, A. van den Berg b, L.J. Nagels a,) a Department of Chemistry, UniÕersity of Antwerpen() RUCA , Groenenborgerlaan 171, B-2020 Antwerp, Belgium b MESA Research Institute, UniÕersity of Twente, P.O. Box 217, 7500 AE Enschede, Netherlands c KluyÕer Laboratory for Biotechnology, Delft UniÕersity of Technology, Julianalaan 67, 2628 BC Delft, Netherlands Received 14 February 2000; received in revised form 12 May 2000; accepted 15 May 2000

Abstract

An ion-selective field effect transistorŽ. ISFET was applied as a potentiometric detector in liquid chromatography Ž. LC for the determination of organic acids. The ISFET was prepared by coating the gate insulator of the encapsulated transistor with a polyŽ vinyl chloride.Ž PVC . matrix membrane containing methyltridodecylammoniumchloride, which enables the detection of organic anions. The ISFET was tested for its applicability as detector for carboxylic acids in ion-exchange and reversed-phase chromatography. Its analytical characteristics were compared to those of a coated-wire electrodeŽ. CWE and of a conventional type of ion-selective electrode Ž. ISE . q 2000 Elsevier Science S.A. All rights reserved.

Keywords: ISFET; Liquid chromatography; Potentiometric detection; Carboxylic acids

1. Introduction conductivity detection may be limited by a large back- ground signal. Therefore, a suppressor system is usually In liquid chromatographyŽ. LC , several systems are used in combination with a conductivity detector to reduce used routinely for the determination of organic acids, eluent conductivity. Potentiometric detection can be an including reversed-phasewx 1,2 , ion-exchange wx 3 , ion-ex- interesting alternative in these cases. It is a relatively clusionwx 2,4 and ion-pair wx 5 chromatography. Also, capil- simple and inexpensive detection method. A distinct ad- lary electrophoresisŽ. CE has proved to be a successful vantage of potentiometric detectors is that their response is technique for the separation and quantification of short- independent on the size of the electrode that makes them chain carboxylic acidswx 6 . well suited for use in combination with micro-separation In this study two chromatographic separation methods, techniques such as CE. Still, potentiometric sensors are not namely reversed-phaseŽ. RP and ion-exchange were ap- yet routinely applied in LC or in CE. Only a limited plied. UV- and conductivity detection are most commonly number of research groups work on this subject. Simon et employed in combination with these systems. However, al. applied liquid-membrane-based potentiometric detec- for carboxylic acids, which lack chromophoric groups, low tion in LCwx 7 and in CE wx 8 . Chen et al. employed a UV detection is necessary. This implies a loss in sensitiv- metallic copper electrodewx 5,9 and a tungsten oxide elec- ity and the occurrence of matrix interferences, especially trodewx 10 for the LC determination of organic acids. In the when complex samples are analysed. The sensitivity of work of Hauser et al., different classes of organic ions were detected in CE, at a metallic copper electrodewx 11 and at coated-wire electrodesŽ. CWEswx 12 . Isildak wx 13 recently used an all solid-state contact tubular PVC-matrix ) Corresponding author. Fax: q32-3-2180233. E-mail address: [email protected]Ž. L.J. Nagels . membrane electrode for the detection of anions in ion- 1 On leave from University ``Politehnica'' of Bucharest, Bucharest, chromatography. In our laboratory we applied liquid-mem- Romania. brane electrodeswx 14,15 and conducting polymer coated

0925-4005r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S0925-4005Ž. 00 00531-1 I. Poels et al.rSensors and Actuators B 67() 2000 294±299 295 electrodeswx 16,17 for the detection of organic acids in LC consisted of 0.1 M KCl containing 10y3 M tetrapentylam- and CE. moniumchloride. Ion-selective field effect transistorsŽ. ISFETs are mainly To prepare the CWEs, the membrane mixture was employed in static measurements, comparable with the deposited directly onto a glassy carbonŽ. GC electrode. conventional way of using ion-selective electrodesŽ. ISEs . Before coating, the GC electrodesŽ. 3 mm diameter , However, the use of ISFETs in flow-through and flow-in- mounted in plastic bodies, were polished with a 5 mm-grid jection analysisŽ. FIA systems has also been widely docu- polishing sheet and cleaned with water and ethanol. Three mentedwx 18,19 . As far as we know, only one paper reports layers were deposited consecutively on the electrode by the use of an ISFET in chromatographywx 20 , namely for using a Pasteur pipette. The THF was allowed to evaporate the determination of alkali-metal cations in ion-chromatog- at room temperature for at least 2 h. raphy. In the present study, we examined the possibility of Ion-selective membranes for ISFETs were formed by using an ISFET-based anion sensor as a potentiometric casting three consecutive drops of the membrane cocktail detector for organic acids separated using two liquid chro- onto the gate area by using a Pasteur pipette. The mem- matographic methods: ion-exchange and reversed-phase. brane was dried for at least 2 h at room temperature. The analytical characteristics of the ISFET were compared with those of a CWE and of a conventional type of ISE 2.3. ISFET with internal reference solution. The ISFET chips used in the present study were devel- oped and produced at the MESAq Research Institute of 2. Experimental the University of TwenteŽ. Enschede, The Netherlands . The chip had a total size of 3=4 mm. The ISFET was an 2.1. Reagents n-channel depletion mode FET, having as gate insulator a

layer of SiO225Ž. 75 nm thick , covered by a layer of Ta O All the chemicals used were of analytical reagent grade. of 125 nm thick. The gate area had a size of 15=500 mm. Eluents were prepared daily and were filtered through a For details of the fabrication sequence of the ISFET, see 0.2 mm membrane filter and degassed with helium before Ref.wx 21 . The ISFET chips were mounted onto a cylindri- use. The eluent used in ion-exchange chromatography was cal plastic body and encapsulated, except for the gate prepared by dilution of a 50%Ž. wrw NaOH solution with region, using epoxy resinŽ Hysolw epoxy obtained from low carbonate concentration, obtained from J.T. Baker. Dexter Electronics Materials Division NY. . The ISFET This eluent was kept under a helium atmosphere during the chips were placed in such a way that the gate region was analysis to avoid carbonate contamination. Organic acids in the centre of the plastic body. were purchased from Merck, UCB, Acros, CRB, Aldrich, Fluka, Sigma and LBC. Stock solutions of fumaric acid 2.4. Apparatus and of organic acid mixtures were prepared in deionised water. Dilutions of the stock solutions in the running The LC-system comprised a SP8810 isocratic pump eluent were made daily and were filteredŽ. 0.2 mm before Ž.ŽSpectra Physics, San Jose, CA and a Valco injector 50 injection. ml loop. . For ion-exchange chromatography, a pellicular anion-exchange columnŽ IonPac AS11, 250=4 mm i.d., 2.2. Ion-selectiÕe membranes Dionex, Sunnyvale, CA. was used in series with a guard columnŽ. Dionex IonPac AG11, 50=4 mm i.d. . A RP-8 The composition of the anion-selective membrane was columnŽ. Merck Lichrospher RP-8; 5 mm, 125=4 mm i.d. 6% methyltridodecylammonium-chloride, 65% o- was used for reversed phase chromatography. For mea- nitrophenyl octyl ether and 29% polyŽ. vinyl chloride surements, the ISFETs, CWEs and ISEs were placed in a Ž.PVC . 350 mg of the membrane material was dissolved in home-made large-volume wall-jet type flow cellwx 22 . A 3.5 ml tetrahydrofuranŽ. THF and this solution was used schematic representation of the flow cell with mounted directly after mixing. All membrane components were ISFET is given in Fig. 1. The column effluent was directed obtained from FlukaŽ. Buchs, Switzerland . perpendicularly towards the ion-selective membrane. The Membranes for conventional ISEs were prepared by distance from the LC tubing-outlet to the ion-selective pouring the membrane cocktail into a glass ringŽ 30 mm membrane was 100 mm. An Orion 800500 Ross electrode diameter. , which was placed on a glass plate and dried at was used as reference electrode. The response of the room temperature for at least 24 h. After the membrane ISFETs was measured with a source and drain follower was peeled off from the glass plate, a disk membrane was type of ISFET amplifierŽ Sprenkels Consultancy, Lelystad, cut out and glued onto a PVC tubeŽ. 3 mm diameter . The The Netherlands. in a constant drain-current mode, with a other end of the PVC tube was connected to a disposable constant drain-source potentialŽ. 0.5 V . The output signal 5-ml syringe. A silverrsilver chloride wire was used as an was recorded unamplified on a PC 1000 data acquisition internal reference electrode. The internal filling solution system from Thermo Separation ProductsŽ. San Jose, CA . 296 I. Poels et al.rSensors and Actuators B 67() 2000 294±299

for the electrode used in this study were determined by the separate solution method and were published in Ref.wx 24 .

3.1. Ion-exchange chromatography

In anion-exchange chromatography, the acids were separated in their anionic forms using a strongly alkaline NaOH solution as an eluent. In a recent studywx 24 , it was shown that the coating material used in this work allows a sensitive detection of organic acids in this system. This is because the membrane material shows a high selectivity towards lipophilic anionsŽ. Hofmeister behaviour . There- fore, the electrode material is not very responsive to the

Fig. 1. Schematic representation of the flow cell with mounted ISFET.

In the case of the CWEs and ISEs, the membrane potential was measured against the reference electrode using a high impedance amplifierŽ internal resistance 1013 V, Knick, type 87 F. . The signals were amplified 10 times with a home-made amplifier and then recorded by the data acquisition system.

3. Results and discussion

The use of a potentiometric flow-through detector for LC requires the choice of a suitable eluent and an appro- priate membrane coating material. A suitable eluent is an eluent that permits both an efficient chromatographic separation and a sensitive detection. Both the analyte ion i, and the eluent ion j have a contribution to the response of the potentiometric detectors. This response can be given by the Nikolskii±Eisen- man equation:

z i RT z s q q pot j E E( ln ciijjKc zFi ž/ where E( is a constant potential, ciiand z represent the concentration and the charge of the analyte ion, and cj and pot z jijrefer to the eluent ion. K is the selectivity coefficient that describes the response of the sensor for the eluent ion j versus the analyte ion i. A sensitive detection can only be obtained when the concentration of the eluent ion is pot relatively low and if it has a low K ij value. Membrane electrodes based on classical ion-exchangers such as quaternary ammonium compounds generally dis- y) y) y) play the following selectivity series: ClO4 SCN I y) y) y) y; y; 2y; 2y Fig. 2. Chromatograms of a mixture of four carboxylic acids obtained NO3344Br Cl HCO OAc SO HPO wx23 . This sequence is known as the Hofmeister series. It is using three different potentiometric sensor types:Ž. a ISFET, Ž. b CWE andŽ. c ISE. Column: Dionex Ion Pac AS11, 250=4 mm i.d. Flow-rate: dependent on the free enthalpy of hydration of these y 1.5 ml min 1. Injection volume: 50 ml. Eluent: 8 mM NaOH. Injected analyte anions. Therefore, the electrode is more selective concentration: 10y4 M. Peak identification:Ž. 1 pyruvic acid, Ž. 2 malic for lipophilic anions. Potentiometric selectivity coefficients acid,Ž. 3 system peak, Ž. 4 ketoglutaric acid and Ž. 5 fumaric acid. I. Poels et al.rSensors and Actuators B 67() 2000 294±299 297 hydroxyl eluent anion, which is a very hydrophilic anion, but is highly responsive to the organic acid anions. An 8 mM NaOH eluentŽ. pHs12.1 was used to separate a mixture of four carboxylic acids of analytical inter- est. It contained pyruvic, malic, a-ketoglutaric and fumaric acid. The chromatograms obtained with the ISFET, CWE and ISE are shown in Fig. 2. In the case of fumaric acid, the detection limit, determined for a signal-to-noise ratio of 3, was 2=10y6 M for all the three sensors. This corresponds to an injected amount of 100 pmol and is comparable to the value obtained with suppressed conductivity detectionwx 25 . Calibration curves for fumaric acid were measured with the ISFET, CWE and ISE, showing peak heightŽ. in mV versus the logarithm of injected concentrations. They are Ž.` presented in Fig. 3. A logarithmic relationship was ob- Fig. 4. Response time as a function of flow-rate for the ISFET , CWE y Ž.v and ISE Ž.I . Measurements were done in a FIA set-up using the = 5 y served for injected concentrations higher than 5 10 M. chromatographic conditions as in Fig. 1. Concentration plugs of 10 3 M At lower concentrations the curves showed a linear depen- fumaric acid were injected. dence, as predicted by a model described earlierwx 15 . The correlation coefficients in the linear part of the curves, response times of the ISFET and CWE were both around 1 measured with the ISFET, CWE and ISE, were 0.999, s, whereas for the ISE this value was bigger than 2 s. 1.000 and 0.999, respectively.

3.2. Response time measurements

A FIA set-up was used to measure the response times of the ISFET, CWE and ISE. We determined the t90 response wx time, as recommended by IUPAC 26 . t90 is defined as the time required to reach 90% of the signal steady-state value. We injected rectangular concentration pulses of 10y3 M fumaric acid, using a 500 ml injection loopŽ pulse time of 15 s. . Fig. 4 gives the response time t90 as a function of the flow-rate for the three sensors. It reveals that the response time increases when the flow-rate is lowered. This is caused by the increased thickness of the diffusion layer near the membranewx 14 . The three types of potentiometric detectors all show acceptable response times for a conventional LC system. However, both the CWE and ISFET type sensors have a faster response time than the conventional ISE detector. At 1.5 ml miny1, the

Fig. 5. Chromatograms of a mixture of six carboxylic acids obtained using three different potentiometric sensor types:Ž. a ISFET, Ž. b CWE andŽ. c ISE. Column: Merck Lichrospher RP-8, 125=4 mm i.d. Flow-rate: 0.5 ml miny1. Injection volume: 50 ml. Eluent: 1 mM phosphoric acid. Fig. 3. Calibration curves of fumaric acid measured at an ISFET Ž.` ,a Injected concentration: 10y4 M. Peak identification:Ž. 1 tartaric acid, Ž. 2 CWE Ž.v and an ISE Ž.I in anion-exchange chromatography. Condi- malic acid,Ž. 3 lactic acid, Ž. 4 citric acid, Ž. 5 fumaric acid and Ž. 6 succinic tions as in Fig. 1. acid. 298 I. Poels et al.rSensors and Actuators B 67() 2000 294±299

times of the ISFET- and CWE-based systems were also comparable and somewhat superior to the conventional ISE type configuration. Similar calibration curves were also obtained for the CWE and ISFET based systems, showing a linear relationship in the lower part of the curve. Both the ISFET and the CWE systems are interesting detection devices for use in miniaturised systems of analysis such as micro-Total Analysis System Ž.mTAS . The CWE system is very simple, and more robustŽ see Ref. wx24. than the ISFET system where poor adhesion of the coating to the gate part was sometimes observed Ð this should be improved in future work. The ISFET system Fig. 6. Calibration curves of fumaric acid measured at an ISFET Ž.` and allows transport of a low impedance signal to the data a CWE Ž.v in reversed-phase chromatography. Conditions as in Fig. 4. station enabling a high degree of miniaturisation and inte- gration in ``lab on a chip'' or mTAS systemswx 27 .

3.3. ReÕersed-phase chromatography Acknowledgements In reversed-phase chromatography, eluents of low pH are required to separate organic acids. These eluents sup- This research has been partially supported by the Tech- press the ionisation of the acids. In a previous studywx 16 , it nology Foundation STW, applied science division of NWO, was shown that a 1 mM phosphoric acid eluent allowed a Project Biomass no. DST 66.4351. The authors kindly sensitive potentiometric detection of organic acids in this acknowledge Prof. P. Bergveld and J. Bomer of the system, using a conducting oligomer electrode. Chro- Biosensors group, MESAq, University of Twente for matograms were measured under the same conditions for delivering the ISFETs. L. Nagels thanks the FWO for the three sensor typesŽ. see Fig. 5 . The mixture of organic financial support. acids contained tartaric, malic, lactic, citric, fumaric and succinic acid. 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